Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent cloning studies confirm the presence of two subtypes of bombesin (Bn) receptors. In contrast to the gastrin-releasing peptide (GRP)-preferring subtype, which has been widely studied, nothing is known about the cellular mechanisms of the neuromedin B (NMB)-preferring subtype, which occurs widely in the central nervous system and gastrointestinal tissues, partially because of the lack of a cell line with functional receptors. In the present study we have investigated Bn receptors on the rat glioblastoma cell line C-6, reported to contain mRNA of the NMB receptor subtype. Binding of 125I-[D-Tyr0]NMB to these cells was time- and temperature-dependent, saturable, reversible, and only inhibited by Bn receptor agonists or antagonists. For Bn receptor agonists the relative potencies were: NMB (1.7 nM) approximately equal to litorin (3 nM) greater than ranatensin (8 nM) greater than Bn (19 nM) greater than neuromedin C (NMC) (210 nM) greater than GRP (500 nM). These relative affinities were almost identical to those for the NMB receptor subtype on rat oesophageal tissue and for Balb 3T3 cells stably transfected with the NMB receptor subtype. These potencies differed from those for the GRP receptor subtype on rat pancreatic acini [Bn approximately equal to litorin (4 nM) greater than ranatensin, NMC, GRP (15-20 nM) much greater than NMB (351 nM)]. The relative potencies of four different classes of Bn receptor antagonists were compared. Results from C-6 tumour cells agreed closely with those for binding to the NMB receptor subtype on rat oesophageal tissue and in Balb 3T3 cells stably transfected with this receptor, and differed markedly from those for binding to the GRP receptor subtype on rat pancreatic acini. Four Bn receptor antagonists had a higher affinity for the GRP subtype ([D-Phe6]Bn-(6-13)ethyl ester (500 x), [D-Phe6][psi 13-14,Cpa14]Bn- (6-14) (70 x) (where psi 13-14 refers to the replacement of the -CONH- peptide bond between Leu13 and Met14 by -CH2NH2) [psi 13-14,Leu14]Bn, [D-Phe6]Bn-(6-13) propylamide (30 x)] and two had a higher affinity for the NMB subtype on C-6 cells and transfected cells ([D-Pro4,D-Trp7,9,10] substance P-(4-11) (9 x) and [Tyr4,D-Phe12]Bn (18 x)]. In C-6 tumour cells, Bn receptor agonists caused an increase in cytosolic Ca2+ and the generation of inositol phosphates. For both responses, NMB was more than 50-fold more potent than GRP. Neither NMB nor GRP increased cyclic AMP. These results demonstrate that the rat glioblastoma cell line C-6 possesses functional NMB-preferring Bn receptors, and agonist occupation activates phospholipase C, thus increasing cytosolic Ca2+ and inositol phosphate formation. Because the interaction of Bn-related peptides with C-6 cell receptors is identical with that reported in other tissues containing the mRNA for the NMB subtype, this cell line should prove useful in exploring further the cellular basis of action of the peptides that interact with this receptor in the central nervous system and various other tissues.
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PMID:Activation of neuromedin B-preferring bombesin receptors on rat glioblastoma C-6 cells increases cellular Ca2+ and phosphoinositides. 132 46

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
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PMID:Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors. 133 12

Endothelial cells were isolated from rat brain microvessels and grown in vitro. They expressed a high density of [125I-Tyr4]bombesin receptor (Bmax = 0.9 pmol/mg protein) with an apparent Kd value of 10 nM. The pharmacological profile of inhibition of the specific [125I-Tyr4]bombesin binding [bombesin = neuromedin B > gastrin releasing peptide (GRP)] was consistent with the presence of a neuromedin-B-preferring receptor. Addition of bombesin, neuromedin B and GRP increased the activity of phospholipase C as measured by the production of total inositol phosphates and from intracellular Ca2+ measurements. They increase 86Rb+ uptake by the Na+, K+, 2Cl- cotransporter and by a charybdotoxin-sensitive, Ca(2+)-activated K+ channel and 22Na+ uptake by the Na+/H+ exchanger. The pharmacological profiles of activation of phospholipase C, Na+, K+, 2Cl- cotransport and Na+/H+ exchange by bombesin-like peptide were consistent with an involvement of the neuromedin-B-preferring receptor characterized in binding experiments. It is suggested that one of the actions of neuromedin B in brain vessels could be to control K+ secretion by the blood/brain barrier.
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PMID:Properties and functions of a neuromedin-B-preferring bombesin receptor in brain microvascular endothelial cells. 758 82

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus. 779 19

Little is known about the pharmacology or cell biology of human bombesin (Bn) receptors, because they are usually present at low levels and both subtypes are frequently present in the same tissues. Human gastrin-releasing peptide (GRP) receptors (huGRP-R) and human neuromedin B (NMB) receptors (huNMB-R) were stably transfected into BALB/3T3 fibroblasts. Both receptor types were glycosylated, with 35% of the huGRP-R and 38% of the huNMB-R representing carbohydrate residues. The extent of glycosylation of the transfected huGRP-R was the same as that seen in the human glioblastoma cell line U-118. Radiolabeled agonist ligands were rapidly internalized, whereas noninternalized ligand readily dissociated in a temperature-dependent fashion. The affinities of various agonists for binding to the huGRP-R were Bn (Ki = 1.4 +/- 0.2 nM) = 4 x GRP = 300 x NMB. In contrast, affinities for the huNMB-R were NMB (Ki = 8.1 +/- 5.2 nM) = 4 x Bn = 600 x GRP. [F5-D-Phe6,D-Ala11]Bn(6-13)methyl ester was the most potent huGRP-R antagonist, whereas D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2 was the most potent huNMB-R antagonist. Agonist binding to either receptor type caused activation of phospholipase C and increased cellular [3H]inositol phosphate levels. GRP was potent at increasing [3H]inositol phosphate generation in cells expressing the huGRP-R (EC50 = 13.6 +/- 1.3 nM), whereas NMB was similarly potent when acting upon cells expressing the huNMB-R (EC50 = 9.3 +/- 1.4 nM). However, neither receptor type, when stimulated with agonist, caused an increase in cAMP levels. These data show that stably transfected huGRP-R exhibit similar pharmacology for agonists and antagonists, are appropriately glycosylated, and function similarly with respect to their ability to alter biological activity, compared with natively expressed receptors. Minimal native huNMB-R data are available for comparison, but in general the huNMB-R is similar to the rat NMB receptor in its pharmacology and cell biology.
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PMID:Expression and characterization of cloned human bombesin receptors. 783 18

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes. 793 30

The receptor for neuromedin B (NMB-R), a mammalian bombesin-related peptide, is widely distributed in the central nervous system and gastrointestinal tract. While it is known that this receptor is coupled to phospholipase C, like many other phospholipase C-activating receptors, little is known about regulation of the NMB-R subsequent to agonist stimulation. We studied both native NMB-R on C-6 rat glioblastoma cells and wild type NMB-R cloned from rat esophageal muscle which was stably transfected into Balb/3T3 fibroblasts. Both cell types rapidly increased [3H]inositol phosphates and [Ca2+]i in response to 1 microM NMB, whereas preincubation with 3 nM NMB for 3 h markedly attenuated the ability of 1 microM NMB, but not 1 microM endothelin-1, to alter either cell type's biological activity. Prolonged exposure to 3 nM NMB caused a rapid decrease in the number of NMB-R, with the maximal receptor down-regulation seen at 24 h due to NMB-R internalization. After maximal down-regulation, removal of agonist resulted in a rapid restoration of NMB-R to the cell surface of both cell types. NMB-R recovery at 6 h was blocked by monensin, an inhibitor of receptor recycling, but was not affected by cycloheximide, a protein synthesis inhibitor. Resensitization to agonist paralleled the recovery of NMB-R in both cell types, and resensitization likewise was blocked by monensin. Our data demonstrate that the NMB-R undergoes rapid homologous desensitization consequent to agonist stimulation, which is mediated by receptor down-regulation and which, in turn, is regulated by internalization. During resensitization, NMB-R reappearance on the cell surface membrane is independent of protein synthesis and is due to a recycling from an intracellular site.
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PMID:Desensitization of neuromedin B receptors (NMB-R) on native and NMB-R-transfected cells involves down-regulation and internalization. 816 69

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.
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PMID:GRP-preferring bombesin receptors increase generation of inositol phosphates and tension in rat myometrium. 827 18

Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.
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PMID:Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes. 838 41

Mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) are regulatory neuropeptides involved in numerous physiologic processes, and have been implicated as autocrine and/or paracrine growth factors in human lung carcinoma. Three structurally and pharmacologically distinct bombesin receptor subtypes have been isolated and characterized: the gastrin releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). The three receptors are structurally related, sharing about 50% amino acid identity. They are members of the G-protein coupled receptor superfamily with a seven predicted transmembrane segment topology characteristic of receptors in this family. The signal transduction pathway for GRP-R and NMB-R involves coupling to a pertussis-toxin insensitive G-protein, activation of phospholipase C (PLC), generation of inositol trisphosphate (IP3), release of intracellular calcium, and activation of protein kinase C. While all three bombesin receptors are activated by bombesin agonists, GRP-R, NMB-R, and BRS-3 have very different affinities for the mammalian bombesin-like peptides GRP and NMB, as well as bombesin receptor antagonists. The three bombesin receptor subtypes are expressed in an overlapping subset of human lung carcinoma cell lines. Any therapeutic strategy based on modulation of bombesin growth responses in human lung carcinoma would be well served to take into account the pharmacologic heterogeneity of the relevant receptors.
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PMID:Bombesin receptor structure and expression in human lung carcinoma cell lines. 880 6


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